Calcium is the fifth most common element in the body where 99% exists in the bones as crystalline hydroxyapatite. The extracellular fluids contain about 0.1% of the total body calcium and of the extracellular fluids, about 30% exists in the blood plasma.
The physiological functions of calcium are diverse. Intracellularly calcium modulates the activities of several enzymes, most notably adenylate cyclase and calmodulin. It is also involved in the regulation of a multitude of cellular functions including fertilization, mitosis, cell motility and ancillary action. In striated muscle, calcium activates contraction of the musin fibrils through combination with troponin a calcium binding protein. Calcium also serves to regulate membrane permeability, causes neurotransmitter release and diminishes neuromuscular excitability. For an in-depth discussion of calcium metabolism and function see Fundamentals of Clinical Chemistry, 3rd ed., Editor Norbert W. Tietz, W. B. Saunders Co. (1987).
Clinically, serum calcium levels are of significant diagnostic value. The reference range is very narrow, 2.20 to 2.55 mmol/L, and slight deviations above or below these levels are diagnostic of several physiological disorders. The two most common diseases associated with hypercalcemia (elevated serum calcium) are hyperparathyroidism and malignancy, especially when the malignancy has metastasized to the skeleton and caused bone destruction. Decreased serum calcium levels (hypocalcemia) is commonly associated with hypoparathyroidism. In newborn infants about 1% have significant hypocalcemia (serum calcium <1.75 mmol/L) and exhibit symptoms of hypocalcemia which include irritability, twitching and convulsions which require immediate medical intervention.
Magnesium, like calcium, is one of the major elements found in the body. A typical 70 kg human adult contains about 20 to 28 g of magnesium of which about 55% is found in the bones and 27% in the muscles. The serum reference range of magnesium is also rather narrow being from 0.65 to 1.05 mmol/L. Low levels of serum magnesium, hypomagnesemia (<0.5 mmol/L) is manifested by impairment of neuromuscular function which leads to hyperirritability, tetany and convulsions, symptoms which are nearly identical to hypocalcemia. Increased serum magnesium levels have a sedative effect on the body.
Given the nearly identical clinical symptoms of low serum calcium and low serum magnesium, it is imperative to delineate which element is causing the clinical symptoms. Often both serum calcium and magnesium measurements are necessary to determine which element or if both elements are low.
The reference method for measuring calcium and magnesium is atomic absorption. The technique is nearly interference free, requires a small sample volume and gives good precision and reproducibility. For routine measurements, atomic absorption is somewhat inconvenient, requires expensive instrumentation and a rather skilled operator to perform assays.
Present methodologies in routine use in clinical laboratories for measuring calcium use procedures based on ortho-cresolphthalein complexone (CPC) and arsenazo III. Although both methods are in wide use, each is not without its drawbacks. The sensitivity of CPC methods is very dependent on pH. For maximum sensitivity the reaction is carried out at a pH of about 11.7. At these alkaline pH values, however, the reagent readily absorbs ambient carbon dioxide which combines with water to form carbonic acid which gradually reduces the reagent pH and eventually renders the reagent non-functional for calcium measurements. Also, CPC is rather non-selective and binds magnesium and other heavy metals. To eliminate magnesium interference at the levels normally encountered in serum, 8-hydroxyquinoline is added to chelate magnesium, but this compound also chelates calcium and decreases the sensitivity by 25 to 40%. Arsenazo III methods do not suffer from the problems of high pH and magnesium interference (depending on measurement pH) inherent in the CPC methods. It binds calcium under weakly acidic conditions, e.g. pH 5 to 6, and if the calcium measurement is made at a pH less than 7, binding of magnesium is negligible. Although arsenazo III eliminates many of the disadvantages of CPC methods, it suffers from rather low sensitivity and environmental concerns. Each mole of arsenazo III contains 2 moles of arsenic, and disposal of the arsenazo III reagents is becoming a serious issue in many countries due to concerns of contamination of the water supply with arsenic.
Tanaka, et al. (U.S. Pat. No. 4,966,784) and Kaufman et al. (U.S. Pat. No. 5,589,348) have developed methods for measuring calcium using chlorophosphonazo III. Although this chromophore does not contain arsenic, it tends to have relatively a high reagent blank absorbance which limits the linearity for calcium on many analyzers. Chapoteau et al. have developed calcium methods using phenolic derivatives of tetraacetic acid (U.S. Pat. No. 5,262,330). However, to bind calcium the assays still require an alkaline pH.
The problems plaguing calcium assays are also common to magnesium assays. Calmagite methods (U.S. Pat. No. 4,383,043) are routinely used by many clinical laboratories, and other methods have been developed using Xylidyl Blue, Xylazo Violet I and II (U.S. Pat. No. 4,503,156), and Erichrome Black T (U.S. Pat. No. 4,383,043). As with calcium assays, all the preceding magnesium methods require a high pH (>>9) and pH stability of the reagent in an uncapped vial is limited due to absorption of ambient carbon dioxide. A recent method for measuring magnesium was published using chlorophosphonazo III (U.S. Pat. Nos. 5,589,348 and 5,397,710). Although this chromophore overcomes the high pH requirement to bind magnesium, it still suffers from a relatively high reagent blank absorbance which limits the linearity for magnesium on many clinical chemistry analyzers used in clinical laboratories.
Thus, there are unmet needs for methods to quantitatively measure calcium and magnesium in analytical samples. The method should a) bind calcium and magnesium around a neutral or slightly acidic pH, b) the chromophore should contain no toxic elements e.g. arsenic, c) the chromophore should have a relatively low reagent blank absorbance, and d) the reagent should have safe handling characteristics, e.g. a pH around neutrality, in case of skin contact or spillage.
Sodium is by far the most prevalent cation in the extracellular fluid and in plasma and serum. The main function of sodium in the body is to maintain the normal distribution of water and the osmotic pressure in the extracellular compartment.
Sodium in body fluids, e.g. serum and plasma, is typically measured by either flame emission spectroscopy or sodium ion specific electrodes. Although both methods generally work quite well each is not without its drawbacks. In the United States, OSHA (Occupational Safety and Health Administration) has dictated flame photometers use propane as the fuel. Propane gas leaks can readily occur from tanks, valves and fittings and discharge propane into the work area thus posing a potential explosion hazard. Also flame characteristics may change as the propane tanks reach exhaustion and this may require more frequent calibration or a flame that has unusable characteristics.
Sodium ion specific electrodes overcome the safety issues with flame photometry. Although the electrodes generally work well, they need frequent cleaning to remove protein build-up and they have a finite working lifetime and electrode replacement cost is somewhat expensive. Also the initial cost of the instrumentation to run the electrodes on is prohibitive for many small clinical laboratories.
Some attempts have been made to measure sodium colorimetrically. Chapoteau et al. (U.S. Pat. No. 4,808,539) and Cram et al. (U.S. Pat. No. 5,011,924) have respectively developed procedures using “chromogenic cryptands” and “chromogenic cryptahemispherands” to measure sodium in serum. Also a kinetic enzymatic procedure was published by Berry et al. (Clin. Chem. 34, 2295-2298 (1988)) using the enzyme beta-galactosidase where the enzyme activity was activated in the presence of low concentrations of sodium.
In 1966 Budesinsky et. al. (Tschechoslow. Pat. Nr. 122379) described the synthesis of a series of chromotroptic acid derivatives including phosphonazo III and presented spectral data of chelates of several heavy metals and transition metal ions (Coll. Czech. Chem. Comm. 32, 1967 and Talanta 15(10), 1063-4, 1968). A summary of the data was later published in Chelates in Analytical Chemistry, 1969, Vol. 2, Marcel Dekker Inc., New York, N.Y. (p1-91). About the same time, a group of Russian scientists presented spectral data of phosphonazo III and other derivatives with several divalent and transition metal ions (Luken et al. Dokl, Akad. Nauk. SSSR 173(2), 361-363, 1967) and other investigators demonstrated the complexation and spectral properties of several rare earth metals with phosphonazo III (Zh. Anal. Khim., 26(4), J, 772-6, 1971; Zh. Anal. Khim. 32(4), 674-678, 1997; and Tr. Vses. Nauch.-Issled. Inst. Khim. Reaktivov Osobo Chist. Khim. Veshchesestv, 1967, No. 30, 42-9).